Continued study of the human genome and more recently the mouse genome has resulted in a clear need for sorting large numbers of chromosomes. The ability to identify, isolate and amplify specific genetic sequences is pivotal to genetic mapping, the search for DNA sequence probes, construction of chromosome-specific libraries, identification of disease genes, and understanding the basic genome organization. Since the human genome is comprised of 3 billion DNA base pairs distributed over 24 chromosome types, identification of a specific gene or gene mutation in this system is a complex undertaking. Chromosome sorting can be used to narrow the source of the DNA to a specific chromosome. By sorting human chromosomes prior to genetic mapping analysis, the complexity of the study is reduced by a factor of about 24. Sorted chromosomes have proven to be invaluable to the National Laboratory Gene Library Project for the construction of small insert libraries (up to 25 kb) prepared from bacteriophages and large insert libraries (35 kb to several hundred kb) prepared from cosmids and yeast artificial chromosomes (YACs).
Analysis of a single chromosome in a flow cytometer was first reported in 1975. Initially, a single fluorescent DNA probe was used to distinguish between chromosome types; however, by 1980, two-fluorochrome analysis of human chromosomes had been developed making it possible to resolve 20 of the 24 types of human chromosomes. Improvements in buffers, high speed sorting, slit scan analysis and data analysis, were subsequently made.
Chromosome sorting is currently achieved using commercial flow cytometers which rely on droplet sorting. Briefly, individual chromosomes stained with one or two fluorescent dyes pass through either one or two sequential focused laser beams tuned to excite the fluorescent stains. Optimally, each chromosome has a unique staining pattern allowing identification based on distinctive fluorescent characteristics such as fluorescence intensity. A liquid carrier stream is caused to break into droplets containing chromosomes. Droplets containing chromosomes of interest are charged. All droplets pass through an electric field and the charged droplets are deflected into a separate collection tube, thus sorting the chromosomes. See, e.g., "Flow Sorters for Biological Cells," by Tore Lindmo et al., Flow Cytometry and Sorting, Second Edition, Wiley-Liss, Inc.: New York, 1990; pages 145-169.
While flow cytometry is currently the principal technique used in sorting chromosomes, drawbacks persist; separation rates are limited by the rate of stable droplet formation. The degree of separation is inversely proportional to analysis rate, that is, the faster the sorting, the lower the purity. For example, in sorting chromosome no 4 of a hamster-human cell line (UV20 HL21-27), the purity is found to drop from 98 to 91% when the analysis rate is increased from 5000 to 18,000 chromosomes s.sup.-1. Furthermore, in order to obtain high purity chromosomes, the probability of having more than one particle per drop must be extremely low; hence, particles are processed at approximately one-tenth the rate of droplet formation. Typical commercial sorters have analysis rates of approximately 1500 chromosomes s.sup.-. This translates into sort rates of less than 50 chromosomes s.sup.-. At this rate, about 60 hours of sorting time is required to obtain 1 .mu.g of DNA. Sorting requirements for YAC cloning often require continuous around the clock sorting for as much as two weeks.
It is possible to achieve rates of the order of 200,000 droplets s.sup.-1 in state-of-the-art flow cytometers, which translates to chromosome analysis rates in excess of 20,000 chromosomes s.sup.-1. To achieve such high droplet rates, however, the sorter must operate at high pressure (200 psi) resulting in sorting instability and random chromosome damage. By reducing the pressure to about 120 psi, droplets can be produced at rates of 140,000 s.sup.-1, improving sorting stability and reducing DNA degradation, but at the cost of lower chromosome throughput.
Methods of chromosome separation not relying on droplet formation would overcome the limitations associated with traditional sorting. In U.S. Pat. No. 4,395,397 for "Apparatus And Method For Killing Unwanted Cells," which issued to Howard M. Shapiro on Jul. 26, 1983, the removal of a subpopulation of unwanted cells from a flowing liquid stream containing a suspension of living cells is described. After detecting the presence of the unwanted cells, such cells are destroyed using a high-power laser. In "High-Speed Photodamage Cell Selection Using Bromodeoxyuridine/Hoechst 33342 Photosensitized Cell Killing," by Hans Herweijer et al., Cytometry 9, 143 (1988), the authors describe a photodamage cell sorter where unwanted cells detected by a first laser are destroyed by radiation from a second laser to which the unwanted cells are photosensitive. The radiation from the second laser is switched on and off (to permit desired cells to proceed unharmed through the apparatus) using an acoustoopic crystal. Cells are made photosensitive by growing the cells in the presence of 5-bromo-2'-deoxyuridine and staining with Hoechst 33342. A 400 mW ultraviolet light source is employed which permits 30,000 cells s.sup.-1 to be sorted. Subsequent work in "High-Speed Photodamage Cell Selection Using A Frequency-Doubled Argon Ion Laser," by Jan F. Keij et al., Cytometry 19, 209 (1995), overcame the extreme photosensitivity of the stained cells and the 2-3 day culturing process for attaching the photosensitizer to the cells, by exploiting the intrinsic photosensitivity of the cellular DNA. Part of the far ultraviolet direct DNA damage is due to the production of thymidine dimers which hinder DNA replication in the cells. The short lifetime of the frequency-doubling crystals employed expected to be overcome using large argon ion lasers capable of emissions at 257-275 nm. In "The Role Of DNA Damage In PM2 Viral Inactivation By Methylene Blue Photosensitization," by Kathleen G. Specht, Photochem. and Photobiol. 59, 506 (1994), viral inactivation was observed as a result of phototreatment of viruses in which phenothiazines including methylene blue, toluidine blue and azure B were bound to nucleic acids in vitro.
TOTO and YOYO have been utilized for DNA analysis in flow cytometry. See, e.g., "TOTO and YOYO: New Very Bright Fluorochromes For DNA Content Analyses by Flow Cytometry," by G. T. Hirons et al., Cytometry 15, 1 (1994), "Stable Fluorescent Complexes Of Double-Stranded DNA with Bis-Intercalating Asymmetric Cyanine Dyes: Properties And Applications," by H. S. Rye et al., Nucleic Acids Res. 20, 2803 (1992), "Stable Dye-DNA Intercalation Complexes As Reagents For High-Sensitivity Fluorescence Detection," by Alexander N. Glazer and Hays S. Rye, Nature 359, 859 (1992), "Rapid Sizing Of Individual Fluorescently Stained DNA Fragments By Flow Cytometry," by Peter M. Goodwin et al., Nucleic Acids Research 21, 803 (1993), and "Characterization Of DNA Size Determination Of Small Fragments By Flow Cytometry," by Jeffrey T. Petty et al., Anal. Chem. 67, 1755 (1995). However, the ability of TOTO and YOYO to render the intercalated DNA unclonable has not been reported.
In "Development Of A Plaque Reduction Assay And Application To The Study Of Psoralen-Damaged DNA," by Kathy E. Yokobata et al., Photochem. and Photobiol. 43, 391 (1986), the photochemically induced reaction of a psoralen derivative with bacteriophage DNA is found to reduce the infectivity of the covalently modified DNA. This procedure is conducted in a bulk sample, and no separation of DNA fragments is taught or suggested.
Accordingly, it is an object of the present invention to provide a method for rapidly, but accurately selecting and collecting chosen strands of DNA from a mixture of DNA strands without requiring high-power, far-ultraviolet lasers and expensive dye removal steps.
Another object of the invention is to provide an apparatus for rapidly, but accurately rendering selected strands of DNA from a mixture of DNA strands unclonable without requiring high-power, far-ultraviolet lasers.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.